Study on Mechanism of Bushen Huoxue Recipe(补肾活血方)Promoting Osteogenesis of BMSCs and Preventing and Treating Osteoporosis in Rats
Objective Studies have shown that microRNA(miRNA)and Kruppel-like factor 10(KLF10)regulate the pro-gression of osteoporosis.The purpose of this study was to explore the mechanism of Bushen Huoxue Recipe(补肾活血方)/miR-26b/KLF10 signal regulating bone marrow mesenchymal stem cells(BMSCs)differentiation.Methods The BMSCs of rats were identified and separated.Different doses of Bushen Huoxue Recipe were used to treat BMSCs,and the proliferation effect was detected.Alkaline phosphatase activity and alizarin red staining were used to detect the osteogenic differentiation potential of BMSCs.The qPCR was used to detect the expressions of miR-26b and KLF10 in BMSCs treated with different doses of Bushen Huoxue Recipe.MirtarBase database was used to detect the binding site of miR-26b on the 3'UTR(untranslated region)of KLF10mRNA,and double luciferase report test was used to detect the effect of miR-26b on the activity of KLF10 promoter.The qPCR was used to detect the effect of miR-26b/KLF10 signal on the expressions of BMSCs proliferation and osteogenic differen-tiation markers.Western blot was used to examine whether the Wnt/β-catenin signal was activated.Results Fluorescence-ac-tivated cell sorting analysis showed that most of the isolated primary cells were BMSCs.Bushen Huoxue Recipe significantly pro-moted the proliferation of BMSCs,significantly increased the level of alkaline phosphatase in BMSCs and the potential of osteogen-ic differentiation.Bushen Huoxue Recipe promoted the expressions of miR-26b and KLF10 in BMSCs.There was an miR-26b binding site on the 3UTR(untranslated region)of KLF10 mRNA.In BMSCs,miR-26b inhibited the activity of KLF10 3UTR luciferase.In addition,miR-26b also inhibited the expression of KLF10.CCK8 showed that KLF10 promoted the proliferation of BMSCs,while qPCR showed that miR-26b significantly inhibited the expressions of osteogenic differentiation markers.KLF10 can reverse the inhibitory effect of miR-26b,suggesting that miR-26b/KLF10 signal mediates the effect of Bushen Huoxue Recipe on the proliferation and osteogenic differentiation of BMSCs.Bushen Huoxue Recipe promoted osteogenesis by activating Wnt/β-Catenin signal pathway.Conclusion The effect of Bushen Huoxue Recipe on osteogenesis of BMSCs is regulated by miR-26b/KLF10/Wnt/β-catenin signal axis.
万方数据
维普
